Vaccines have been used for many years to protect humans and animals against a wide variety of infectious diseases. Such conventional vaccines consist of attenuated pathogens (for example, polio virus), killed pathogens (for example, Bordetella pertussis) or immunogenic components of the pathogen (for example, diphtheria toxoid). Some antigens are highly immunogenic and are capable alone of eliciting immune responses. Other antigens, however, fail to induce, for example, a protective immune response or induce only a weak immune response.
Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves.
Immunostimulatory agents or extrinsic adjuvants have been used for many years to improve the host immune responses to immunogenic compositions including vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum. While the usefulness of alum is well established for some applications, it has limitations. For example, it is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response. The antibodies elicited by alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse, which may not be optimal for protection by some vaccinal agents.
Furthermore, studies in rats have demonstrated that alum acts as an IgE adjuvant (ref. 1—Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which the invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the Claims. The disclosure of other references are incorporated by reference into the present disclosures. Studies with tetanus and diphtheria toxoid vaccines also indicate that alum adsorption of vaccines induces IgE antibodies in humans (refs. 2, 3, 4). Therefore, although the inclusion of an aluminum salt in a vaccine formulation may improve its immunogenicity and potency, the fact that it can induce local granulomas and IgE antibodies which may contribute to hypersensitivity reactions warrants careful examination of the practice of alum-adsorption of vaccines for human and animal use.
Some characteristics of desirable adjuvants include:
(1) a lack of toxicity;
(2) an ability to stimulate a long-lasting immune response;
(3) simplicity of manufacture and stability in long-term storage;
(4) an ability to elicit both cellular and humoral immune responses to antigens administered by various routes, if required;
(5) synergy with other adjuvants;
(6) a capability of selectively interacting with populations of antigen presenting cells (APC);
(7) an ability to elicit appropriate TH1 or TH2 cell-specific immune responses;
(8) an ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens; and
(9) that they do not contribute to hypersensitivity reactions.
Of relevance to the present invention is a discussion of the development of pertussis vaccines presented below.
Thus, pertussis or whooping cough is a serious respiratory disease caused by the infection of the respiratory tract by the gram negative organism Bordetella pertussis. Pertussis is a major cause of childhood morbidity and is implicated in 360,000 deaths annually (ref. 5). The most effective method of control of the spread of the disease has proven to be the use of widespread immunization programs. The whole cell pertussis vaccine which was shown to have clinical efficacy in the 1950's, has been effective in controlling pertussis epidemics (refs. 6, 7, 8). The value of the vaccine was illustrated when Japan, Sweden and Great Britain abandoned routine childhood pertussis immunization. Shortly thereafter, these countries experienced major epidemics of pertussis (refs. 9, 10, 11, 12).
Although the whole cell pertussis vaccine is effective in preventing the incidence and spread of disease, the acceptance and uptak of the vaccine has been limited due to reports of vaccine associated adverse effects. Therefore, an impetus for the creation of a non-reactogenic, effective and well defined acellular component pertussis vaccine was created. One of the key features of the acellular vaccine is the chemically detoxified pertussis toxin (PT) component. The presence of native pertussis toxin in the whole cell vaccine has been a source of concern as studies in animal models have shown that it can induce lymphocytosis, histamine sensitization, potentiation of anaphylaxis and IgE antibodies, enhancement of insulin secretion and many other systemic effects (ref. 13). The acellular pertussis vaccines differ with respect to the combinations and quantities of Bordetella pertussis antigens included in the vaccines but the key antigens include the agglutinogens, pertactin, filamentous hemagglutinin (FHA) and pertussis toxin (PT). Although the acellular vaccine has been demonstrated to be immunogenic and of comparable efficacy to the whole cell vaccine, it has not been as effective in preventing bacterial colonization (ref. 14). In addition, the results from a Swedish field trial comparing acellular and whole cell pertussis vaccines indicated that the formaldehyde inactivated pertussis toxin present in the acellular vaccines showed evidence of reversion to toxicity (ref. 15). Therefore, other methods of inactivating the pertussis toxin molecule were required.
To overcome the drawbacks of chemical detoxification, several groups developed genetically detoxified pertussis mutant holotoxin molecules (refs. 16, 17, 18, 19, 20, 21). A promising candidate was the K9G129 mutant. Not only was the immunogenicity of the molecule retained, but the toxicity of this recombinant toxin was greatly diminished (refs. 18, 19, 21). In addition, immunization with the K9G129 mutant stimulated both humoral and cellular pertussis antigen specific responses (ref. 22). Although many clinical trials base the evaluation of the immunogenicity of a vaccine solely on the antibody response following immunization, studies indicate an important role for cellular immunity in protection against this disease. In animal models, the cellular immune response has been demonstrated to be important in the protective response against pertussis as the adoptive transfer of cells from convalescent animals into sublethally irradiated animals conferred protection from challenge with Bordetella pertussis organisms while the passive transfer of immune serum did not (refs. 23, 24). A retrospective study in humans indicated that cell mediated immunity to Bordetella pertussis correlated with a positive history of pertussis (ref. 25). Following natural pertussis infection in humans, both an antibody and cellular immune response are observed (ref. 26). However, immunization with either the whole cell or acellular component vaccines resulted in variable pertussis antigen-specific cellular immune responses (refs. 27, 28). It appeared that the chemical detoxification of the pertussis toxin component destroyed its T cell immunogenicity while the antibody responses were unaffected (ref. 26). Therefore, only the genetically detoxified pertussis toxin molecule could be used to stimulate both a cellular and humoral immune response.
The use of the recombinant PT mutant, K9G129, as a pertussis vaccine component has been well described. A number of different forms of the vaccine have been suggested. Two formulations have been evaluated in humans. The first formulation consisted of 15 μg of the PT mutant which was alum-adsorbed with a total of 0.5 mg of alum per dose (refs. 22, 29) while the other formulation contained 7.5 μg of the K9G129 mutant as well as 10 μg FHA and 10 μg pertactin and was also alum adsorbed (ref. 30). These studies indicated that the genetically detoxified pertussis vaccine candidate was not only safe, immunogenic and could induce a cell mediated response, but, when combined with the FHA and pertactin antigens, it also provided better protection in the intracerebral challenge test than a chemically detoxified component pertussis vaccine (ref. 30). Other suggested formulations include a formaldehyde-treated K9G129 component (ref. 31) and a cellular vaccine derived from a strain of Bordetella pertussis producing the genetically inactivated K9G129 pertussis toxin molecule (ref. 32). The formaldehyde treatment of the K9G129 molecule altered the immunogenicity of the molecule as lower amounts of specific antibodies were induced. The protective ability of the molecule was also decreased as it was less effective in the intracerebral challenge assay (ref. 32). However, the recombinant cellular vaccine derived from the K9G129 producing strain proved to be as effective as the whole cell pertussis vaccine (ref. 32).
Although the preceeding formulations demonstrate the advantages of improved safety and efficacy associated with the use of a genetically detoxified pertussis toxin molecule, they do not address the adverse effects of DPT (diphtheria, pertussis and tetanus) vaccination not associated with the pertussis molecule component (refs. 33, 46). All of the stated formulations involved the use of either 0.3 mg of aluminum phosphate (ref. 32) or 0.5 mg aluminum hydroxide (refs. 29, 30). Aluminum salts were introduced into the DT and DPT vaccine formulations as an adjuvant that would potentiate strong antibody responses when the levels of the toxoids or the numbers of Bordetella pertussis organisms were decreased to avoid adverse reactions (refs. 34, 35) and alum is now routinely used in these vaccines as an adjuvant. However, years of field experience with these adsorbed pertussis vaccines and studies (refs. 36, 37) have demonstrated that, although they contained less of the identified reactogenic vaccine components, local reactions were nonetheless precipitated (refs. 38, 39, 40, 41, 42). Histopathological examination of local abscesses produced following vaccination revealed aluminum hydroxide inclusions in giant cells (ref. 38). Investigation into the frequency of such granulomas indicated that they were associated with the aluminum content in the vaccine as placebo immunized groups which received only the aluminum fraction of the vaccine, exhibited abscess formation at a similar reaction rate (ref. 43). Further evidence in support of the role of aluminum in these local reactions was derived from studies comparing aluminum adjuvant adsorbed and plain cholera and tetanus vaccines (refs. 44, 45). Deep innoculation of the vaccine into the muscle decreases the incidence of these abscesses but although improved techniques can prevent the formation of abscesses (ref. 39), the potentiation of IgE responses by aluminum salts is not affected.
It would be advantageous to provide immunogenic compositions having modulated immune responses to the constituent antigens without the disadvantages of local toxicity and contribution to hypersensitivity of prior art extrinsic adjuvants.